Why working with trypan blue is not a good idea

A review of cell count accuracy and viability using trypan blue

For decades, cells counted under a bright-field microscope have been stained by trypan blue to estimate cell viability. This method relies on the nuclear-exclusion principle: Membrane-impermeable trypan blue is excluded from viable cells but enters permeable dead cells.

Unfortunately, after only a short exposure time of five minutes, trypan blue starts to become toxic to cells1-3. Even viable cells are eventually stained with trypan blue, as when the dye permeates their cell membrane, they die over time (normally between 5 to 30 minutes after exposure).

The toxicity of trypan blue impacts the accuracy of measurements in two ways: (i) the viability analysis changes over time, and (ii) the viability of a cell population is underestimated2.

What are alternative more accurate methods?

Bright-field microscopy is used to validate and quantify cell cultures. It gives ambiguous staining patterns and relies on a series of parameters, including roundness and uniformity, to discriminate cells from non-cellular particles.

Trypan blue stains dead cells in shades ranging from light blue to black, depending on the overall viability of the cell culture.

When using a bright-field microscope, cells that are very lightly stained with trypan blue can be hard to differentiate from unstained cells, and thus hard to identify.

A female scientist is pipetting next to a hemocytometer, trypan blue flask, pipette tip box, tally counter, and notebook.

Also, individual lab members will each have a somewhat subjective view of what is a dead versus a living cell. Therefore, using trypan blue to distinguish between live and dead cells introduces the risk of variation between users and instruments.

For these reasons, fluorometric assays are considered a better choice for the evaluation of cell viability than colorimetric ones, such as trypan blue4.

Because cells have a relatively fixed amount of DNA/RNA, the DNA-interacting fluorophores acridine orange (AO) and 4′,6-diamidino-2-phenylindole (DAPI) will produce a uniform staining pattern for the quantification of cells, compared to the unspecified staining pattern when using bright-field based analysis.

AO is membrane-permeable and stains the total cell population, whereas DAPI only stains the dead cell population. Therefore, fluorometric determination of cell viability using AO and DAPI provides superior accuracy to identify live and dead cells with reduced noise and variation.

Why does safety matter when working with trypan blue?

Ensuring a safe working environment is crucial for businesses. According to the European Chemical Agency (ECHA), trypan blue may cause cancer, genetic defects, and damage fertility, or cause harm to an unborn child5.

Consequently, special precautions should be taken to avoid exposing operators to trypan blue whilst working with and disposing of the dye.

Another important aspect to consider when handling trypan blue is that it is classified as a Group 2B carcinogen5. This makes working with trypan blue potentially hazardous for operators in the lab since exposure should be avoided when possible.

There are safe cell counting alternatives that provide increased precision and improved accuracy.

Close-up of scientist loading a sample into the Via2-Cassette™

Under lock and key in EU laboratories

Recent changes have been made by the ECHA to highlight the fact that trypan blue is toxic to humans, and to restrict its use.

By classifying it as carcinogenic, the aim is to motivate users to phase out trypan blue use in laboratories across Europe5.

As of March 2021, this classification has already resulted in countries like Denmark requiring laboratories to have a permit to use trypan blue. The permit can only be granted if the use of it is strictly necessary in their workflow6,7.

Furthermore, permit-holders are required to limit laboratory professionals’ exposure to trypan blue by ensuring a safe working environment through creating standard operating procedures (SOPs) for all workflows where trypan blue is used.

A part of this is to ensure that all samples and disposables exposed to trypan blue are collected and disposed of separately from normal laboratory waste.

Even though this currently does not apply to countries outside of Europe, it is generally agreed by the scientific community that there are risks associated with trypan blue5, implying that its use should be limited, as it may cause serious harm to users.

What are alternatives to trypan blue?

Alternatives to trypan blue include AO and DAPI which are not carcinogenic8,9. As alternatives to trypan blue, they can support a safer working environment.

Since the Via2-Cassette™ encapsulates these dyes, its design guarantees that any user contact with them is eliminated. This cassette technology can be readily established as a safety measure in laboratories across the world.

The Via2-Cassette™ is a unique microfluidic device that provides unparalleled accuracy and precision of a cell count and eliminates a user’s exposure to chemicals. The Via2-Cassette™ includes a built-in pipette that prevents errors during pipetting and staining.

The fluorescent dyes AO and DAPI are immobilized inside the cassette’s channels, automatically staining the total and dead cell populations, respectively.

During the entire cell counting process, AO and DAPI are kept inside the microfluidic device, significantly reducing the risk of exposure. The Via2-Cassette™ is disposed of after every use, helping to maintain a safe working environment.

The Via2-Cassette™ is used with the NucleoCounter® NC-202™, an automated fluorescent cell counter that provides an analysis of the total and live cells, the dead cell concentration, viability, and the DebrisIndex™ of a sample, all in approximately 30 seconds.

The instruments are constructed using high-quality components that remain stable over time, which together with extensive calibration procedures performed at the time of build, results in low variation between instruments, users and sites. This technology delivers an optimal level of precision to your cell counting.

The NucleoCounter® NC-202™ with the Via2-Cassette™, combines industry-leading accuracy and precision that delivers consistent results while keeping users safe from harm.


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